The Mitochondrial Genome

Avise, John C.  “Footprints of nonsentient design inside the human genome.”  Proceedings of the National Academy of Sciences 107(Suppl 2):8969–8976.  2010.  A paper by a University of California, Irvine evolutionary biologist, written for a 2009 National Academy of Sciences colloquium on the human condition.  It lists features of the human genome that look badly designed, from copying glitches that cause disease to pseudogenes and parasitic mobile elements, and argues that these flaws defy the idea of a caring designer and fit blind evolutionary forces instead.  The article uses it for the charge that mtDNA is badly designed and for its mutation-rate figure.

1. 1a.

   Page 8974:  “Not only is the overall design of mtDNA suboptimal, but it appears downright ludicrous!” ↩

2. 1b.

   Page 8974:  “Why would a wise engineer have put any crucial genes in a caustic cytoplasmic environment in which they are exposed routinely to high concentrations of mutagenic oxygen radicals? Why would he have dictated that the mitochondrial genetic code must differ from the nuclear genetic code, thereby precluding cross-translation between two genomes for which effective communication would seem to be highly desirable?” ↩ ↩ ↩

3. 1c.

   Page 8974:  “Indeed, on a per-nucleotide basis, mtDNA experiences about 5–10 more mutations per unit time than do typical protein-coding nuclear genes (45).”  Reference 45 is Brown, George and Wilson 1979. ↩

4. 1d.

   Page 8974:  “(e.g., little or no genetic recombination, production of a polygenic transcript, limited ability to mend itself, no self-sufficiency in transcription or translation)”. ↩

5. 1e.

   Page 8974:  “Why would a wise designer have imbued mtDNA with some but not all of the genes necessary to carry out its metabolic role (and then put the remaining genes in the nucleus instead)?” ↩

Ainsworth, Claire, and Michael Le Page.  “Evolution's greatest mistakes.”  New Scientist 195(2616):36.  Aug 11, 2007.  A magazine feature with a verdict on each supposed fault in the human body, from lungs to DNA copying.  Its "Burning blueprint" section argues that mitochondrial DNA is badly placed, next to damaging free radicals, and the article quotes that section for its verdict.

1. 2a.

   Section Burning blueprint:  “The process also produces highly damaging molecules called free radicals, so the interior of a mitochondrion is hardly the safest place for vital DNA--and yet it is home to the genes for 13 crucial mitochondrial proteins.” ↩

2. 2b.

   Section Burning blueprint:  “It's a crazy design: like keeping the repair manual for a steam engine by the furnace, where it inevitably becomes charred and unreadable.” ↩

3. 2c.

   Section Burning blueprint:  “The 13 genes cannot simply be moved to the nuclear genome, because then the 13 proteins will be made outside the mitochondria where they are needed. A solution might be to get the mRNA recipes for proteins delivered to the mitochondria, so the genes reside in the nucleus but the proteins are still made inside the mitochondria.” ↩

4. 2d.

   Section Burning blueprint:  “Verdict: If you wanted to build humans to last, mitochondria are the last place you'd put DNA.” ↩

NCBI Reference Sequence.  “Homo sapiens mitochondrion, complete genome.”  GenBank.  Apr 3, 2023.  The reference copy of the human mitochondrial genome in the GenBank database, the revised Cambridge Reference Sequence, with accession NC_012920.1.  The article uses it for the gene counts and for the genetic code that the mitochondrial genes are read with.

1. 3a.

   Section LOCUS line:  “LOCUS NC_012920 16569 bp DNA circular PRI 03-APR-2023”.  Section Features table:  13 CDS, 22 tRNA and 2 rRNA features, each CDS with.  “/transl_table=2”. ↩ ↩

Alexeyev, Mikhail et al.  “The maintenance of mitochondrial DNA integrity—critical analysis and update.”  Cold Spring Harbor Perspectives in Biology 5(5):a012641.  2013.  A review of how mitochondria repair their DNA, written by four researchers at the University of South Alabama.  It marks which points are settled and which are still controversial.  The article uses it for what mitochondria can and cannot repair, and for the point that they can discard damaged copies because they have so many.

1. 4a.

   Section Abstract:  “The process of DNA repair in mitochondria, initially believed nonexistent, has now evolved into a mature area of research.” ↩

2. 4b.

   Section Introduction:  “In contrast, mitochondrial DNA (mtDNA) inheritance is (with a few exceptions) exclusively maternal, and this DNA species is highly redundant, typically a few hundred to a few thousand copies per cell.” ↩

3. 4c.

   Section Introduction:  “Because mtDNA encodes components of four out of five mitochondrial respiratory complexes, it is not surprising that mutations in mtDNA may result in (pathological) alterations in mitochondrial function and mitochondrial diseases”. ↩

4. 4d.

   Section Organization of the mitochondrial genome:  “the mitochondrial genome lacks histones, which has led to the belief that the observed high rate of mtDNA mutagenesis (∼10-fold greater than in nDNA) ... is explained by the lack of “protective” histones. This belief lacks direct experimental support and remains controversial”. ↩

5. 4e.

   Section Nucleotide excision repair:  “There appears to exist a consensus in the literature that substrates of the nucleotide excision repair pathway are not repaired in mtDNA, unless they can be processed by an alternative pathway”. ↩

6. 4f.

   Section Base excision repair:  “Base excision repair (BER) is the predominant and best understood DNA repair pathway in mitochondria”. ↩

7. 4g.

   Section mtDNA Degradation:  “Unlike the nuclear genome, the mitochondrial genome is redundant, consisting of hundreds to thousands of copies per cell. Therefore, a “repair or die” constraint is not imposed on mtDNA.” ↩ ↩

Kukat, Christian et al.  “Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.”  Proceedings of the National Academy of Sciences 108(33):13534–13539.  2011.  A laboratory study of mammalian cells grown in culture, using super-resolution microscopy, which sees structures smaller than ordinary light microscopes can resolve.  The authors measured mitochondrial nucleoids, the protein-wrapped packets that hold mitochondrial DNA, and found a uniform mean size of about 100 nanometres and an average of about 1.4 DNA molecules per packet.  The article uses it to show that the DNA is tightly packed and often holds only one copy.

1. 5a.

   Page 13534:  Abstract.  “Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average ∼1.4 mtDNA molecules per nucleoid).” ↩

2. 5b.

   Page 13535:  “We found on average 2,721 ± 156 copies of mtDNA per human fibroblast when using three different Taqman probes”. ↩

3. 5c.

   Page 13538:  “we report here a much higher abundance and found one TFAM protein molecule per ∼16.6 bp of mtDNA”. ↩ ↩

Kennedy, Scott R. et al.  “Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That Are Inconsistent with Oxidative Damage.”  PLoS Genetics 9(9):e1003794.  2013.  A laboratory study of brain tissue from younger and older people, using Duplex Sequencing, a method that tags each DNA strand so that real rare mutations can be told apart from sequencing errors.  It found that point mutations in mitochondrial DNA rise about fivefold over 80 years of life, and that most of the changes are transitions, the type that copying errors and spontaneous chemical changes produce, not the G-to-T type that oxygen damage produces.  The article uses it because the authors argue that copying errors, not oxygen damage, drive the buildup of mutations.

1. 6a.

   Page 1:  Abstract.  “Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS).”  “Surprisingly, G→T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age.”  “Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.” ↩ ↩

Allio, Remi et al.  “Large Variation in the Ratio of Mitochondrial to Nuclear Mutation Rate across Animals: Implications for Genetic Diversity and the Use of Mitochondrial DNA as a Molecular Marker.”  Molecular Biology and Evolution 34(11):2762–2772.  2017.  A systematic review of published sequence data.  The authors gathered 121 data sets that each hold both mitochondrial and nuclear DNA, plus four large sets, covering 4,676 animal species, and compared how fast each genome changes.  The article uses it to show that the ratio ranges from a few times in insects and spiders to 32.5 per gene in primates, so "several times the nuclear one" understates the human case.

1. 7a.

   Page 2762:  Abstract.  “In nonvertebrates, such as insects and arachnids, the ratio of mtDNA over nuDNA mutation rate varies between 2 and 6, whereas it is above 20, on average, in vertebrates such as scaled reptiles and birds.” ↩

2. 7b.

   Page 2764:  “The mean μmit/μnuc per gene varied from 0.8 in Drosophila and 1.8 in bivalves to 7.4 in birds and 32.5 in primates.” ↩ ↩

Wolfe, Kenneth H. et al.  “Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs.”  Proceedings of the National Academy of Sciences 84(24):9054–9058.  1987.  A comparison of published plant DNA sequences from GenBank and the literature, counting the "silent" changes that do not alter the protein in the mitochondrial, chloroplast and nuclear genomes of flowering plants.  It found that the silent change rate in mitochondrial DNA is less than a third of the chloroplast rate, and the chloroplast rate is only half the nuclear rate.  The article uses it to show that in flowering plants mitochondrial DNA changes more slowly than nuclear DNA.

1. 8a.

   Page 9054:  Abstract.  “Comparison of plant mitochondrial (mt), chloroplast (cp) and nuclear (n) DNA sequences shows that the silent substitution rate in mtDNA is less than one-third that in cpDNA, which in turn evolves only half as fast as plant nDNA.”  “Silent substitution rates in plant and mammalian mtDNAs differ by one or two orders of magnitude, whereas the rates in nDNAs may be similar.” ↩ ↩

Björkholm, Patrik et al.  “Why mitochondria need a genome revisited.”  FEBS Letters 591(1):65–75.  2017.  A 2017 laboratory study that revisits the question its title poses, why mitochondria need a genome.  It expressed all 13 human mitochondrial genes, each with a strong mitochondrial address tag, in the cytosol of human cells, and found that all the proteins except ATP8 went to the endoplasmic reticulum (ER), a membrane network inside the cell.  The article uses it as evidence that protein sorting is a barrier to moving these genes.

1. 9a.

   Section Abstract:  “By expressing all 13 human mitochondrial-encoded genes with strong mitochondrial-targeting sequences in the cytosol of human cells, we show that all proteins, except ATP8, are transported to the endoplasmic reticulum (ER)."  …  "We conclude that subcellular protein targeting constitutes a major barrier to functional transfer of mitochondrial genes to the nuclear genome.” ↩ ↩

Björkholm, Patrik et al.  “Mitochondrial genomes are retained by selective constraints on protein targeting.”  Proceedings of the National Academy of Sciences 112(33):10154–10161.  2015.  A laboratory study that predicted hydrophobic membrane proteins made from mitochondrial DNA would be sent to the endoplasmic reticulum if their genes were moved to the nucleus.  The authors expressed three of them in HeLa cells, found they went to the endoplasmic reticulum, and compared protein domains across bacteria and eukaryotes.  The article uses it for the argument that these genes stay in mitochondria to keep their proteins in the right place.

1. 10a.

   Page 10154:  “One such proposed barrier to functional gene transfers is codon reassignments (12). This explanation has been dismissed because it is not applicable to all mitochondrial genomes.” ↩

2. 10b.

   Page 10154:  “However, this hypothesis has also been rejected because hydrophobic proteins can be imported across the mitochondrial membrane and because some membranespanning proteins are nu-encoded, such as the Lhca and Lhcb proteins in chloroplasts (ct) (14).” ↩

3. 10c.

   Page 10154:  “However, no mitochondrial genes involved in redox regulation have been identified (17). Thus, all hypotheses that have been put forward are controversial and none have been experimentally verified.” ↩ ↩

4. 10d.

   Page 10154:  “In striking contrast to the few proteins encoded by the mitochondrial genome, it is estimated that more than 1,000 mitochondrial proteins are encoded by the nuclear genome (18).” ↩ ↩

5. 10e.

   Page 10155:  “Importantly, it is the hydrophobicity rather than the signal sequence per se that is recognized by SRP.”  SRP is the signal recognition particle, which recognizes proteins headed for the endoplasmic reticulum. ↩

6. 10f.

   Page 10155:  “We conclude that genes for hydrophobic membrane proteins of more than 120 amino acids are likely retained in distinct organelle genomes to ensure a correct localization of these proteins and avoid transport to the ER.” ↩

7. 10g.

   Page 10159:  “the first TMD of the nu-encoded Cox2 protein in legumes is less hydrophobic than the first TMD of the mt-encoded homolog (48)”. ↩

8. 10h.

   Page 10159:  “However, arrest by SRP and subsequent mistargeting to the ER is unlikely to explain all of the peculiarities of mitochondrial genomes in individual species.” ↩

Boominathan, Amutha et al.  “Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant.”  Nucleic Acids Research 44(19):9342–9357.  2016.  A laboratory study that copied the mitochondrial genes ATP8 and ATP6 into the nucleus, gave each a mitochondrial address tag, and put them into cultured cells that carry a patient's mitochondrial mutation and have no working Complex V.  Making ATP8 alone restored only part of the function, but making both let the proteins join Complex V, the enzyme that makes most of the cell's ATP.  The article uses it as the best success, and as one of the two genes moved in cultured cells.

1. 11a.

   Section Abstract:  “Co-expressing both ATP8 and ATP6 genes under similar conditions resulted in stable protein expression leading to successful integration into Complex V of the oxidative phosphorylation machinery.” ↩ ↩

European Medicines Agency.  Withdrawal of application for the marketing authorisation of Lumevoq (lenadogene nolparvovec).  Apr 26, 2023.  The agency's question-and-answer sheet, document EMA/217929/2023, on why the company withdrew its application for approval to sell Lumevoq.  It gives the agency's provisional view that the drug could not have been authorised, because the studies did not show a significant difference in vision.  The article uses it as the regulator's verdict on the ND4 gene therapy: the company withdrew its application on 20 April 2023 while the agency's opinion was against it.

1. 12a.

   Page 1:  “The company withdrew the application on 20 April 2023.” ↩ ↩

2. 12b.

   Page 2:  “At the time of the withdrawal, the Agency had some concerns and its provisional opinion was that Lumevoq could not have been authorised for treating Leber hereditary optic neuropathy caused by the m.11778G>A mutation. The results of the studies did not show a significant difference in vision in eyes injected with Lumevoq and those receiving a sham or placebo injection."  …  "Therefore, at the time of the withdrawal, the Agency’s opinion was that the benefits of Lumevoq did not outweigh its risks.” ↩

Gorman, Gráinne S. et al.  “Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease.”  Annals of Neurology 77(5):753–759.  2015.  A population study that counted adults with mitochondrial disease in North East England at the midyear of 2011, among patients referred to one specialist centre and their families.  It estimated the minimum prevalence (the share of people affected) of mtDNA mutations at 1 in 5,000 adults, a figure the authors give as a minimum.  The article uses it for the cost of mitochondrial disease: at least 1 adult in 5,000 in that region carries a disease-causing mitochondrial mutation.

1. 13a.

   Section Abstract, page 753:  “The minimum prevalence rate for mtDNA mutations was 1 in 5,000 (20 per 100,000), comparable with our previously published prevalence rates.” ↩ ↩

2. 13b.

   Page 754:  The 9.6 figure counts only adults who show symptoms and have a confirmed genetic diagnosis.  “Clinically affected adults with mtDNA mutations (n = 204) were identified, determining a minimum point prevalence of 9.6 in 100,000”. ↩

Milot, Emmanuel et al.  “Mother's curse neutralizes natural selection against a human genetic disease over three centuries.”  Nature Ecology & Evolution 1(9):1400–1406.  2017.  A study of a human genetic disease followed over 290 years in one human population.  Male carriers had low fitness, mostly from high infant mortality, but the analysis predicted a slight, non-significant rise in the mutation's frequency, not the 35.5% per-generation fall expected if mitochondrial DNA came through fathers.  The article uses it as its real-world case of the mother's curse, the idea that mutations which harm only males escape selection.

1. 14a.

   Page 1400, section Abstract:  “Here we provide evidence for the mother's curse in action over 290 years in a human population.”  “Male carriers showed a low fitness relative to non-carriers and to females, mostly explained by their high rate of infant mortality. Despite poor male fitness, selection analysis predicted a slight (albeit non-significant) increase in frequency, which sharply contrasts with the 35.5% per-generation decrease predicted if mitochondrial DNA transmission had been through males instead of females.” ↩ ↩

Demick, David.  “Mitochondria—created to energize us.”  Journal of Creation 20(3):10–12.  2006.  A 2006 article in the Journal of Creation, by David Demick of Creation Ministries International.  It argues that the endosymbiont theory, which says mitochondria began as bacteria taken in by larger cells, is only a hypothesis, and that mitochondria depend on nuclear genes for most of their proteins.  The article quotes it on the genetic code and on the apparent inefficiency of mitochondrial DNA, and it corrects one of its figures.

1. 15a.

   Section Mitochondrial DNA:  “For example, human mitochondria have 83 proteins, but only 13 are coded by mtDNA (mitochondrial DNA).” ↩

2. 15b.

   Section Mitochondrial DNA:  “(On the other hand, from a design standpoint the answer may lie in the simpler protein synthetic machinery served by mtDNA, which uses fewer tRNAs, and is less specific in codon recognition.)” ↩

3. 15c.

   Section Chloroplasts:  “However, viewing mtDNA as inefficient may just be a reflection of our own ignorance of the fine details of mitochondrial function. Deeper knowledge may show that manufacture of certain mitochondrial protein subunits ‘on-site’ is very efficient, just as the energy-harnessing chemistry of the mitochondrial enzymes has been shown to be.” ↩ ↩

Rana, Fazale.  “Why Do Mitochondria Have DNA?”  Reasons to Believe.  Apr 25, 2016.  A web article by Fazale Rana of Reasons to Believe on why mitochondria keep DNA of their own.  It argues that keeping that genome is costly for the cell, that nuclear-made versions of its proteins would be sent to the wrong place, and that the high GC content of its genes makes them durable.  The article quotes it for the cost and for Rana's conclusion that there is a rationale for mitochondrial DNA, and it disputes the GC point.

1. 16a.

   Section Paragraph beginning "Maintaining organellar genomes":  “Maintaining organellar genomes is a costly prospect for the host cell. Typically, a couple hundred proteins are needed to support the production of proteins encoded in mitochondrial genomes.” ↩ ↩

2. 16b.

   Section Numbered point 1:  “If these proteins were encoded in the nuclear genome and produced in the cytoplasm—instead of the lumen of mitochondria—the proteins would be misdirected to the endoplasmic reticulum (ER) instead of the mitochondria.” ↩

3. 16c.

   Section Numbered point 3:  “Since the interiors of mitochondria are harsh environments, the greater GC content of mitochondrial genes serves as an elegant design feature, ensuring their durability in the presence of reactive oxygen species that are by-products of energy metabolism.” ↩

4. 16d.

   Section Paragraph after the numbered points:  “In other words, there is a rationale for why mitochondria have DNA.” ↩ ↩

Elzanowski, Andrzej, and Jim Ostell.  “The Genetic Codes.”  NCBI Taxonomy.  Sep 23, 2024.  Retrieved Sep 27, 2026.  A reference page from the National Center for Biotechnology Information that lists the genetic codes organisms use and how each one differs from the standard code.  It rests mainly on earlier reviews rather than new experiments.  The article uses its vertebrate mitochondrial table for the four codons that differ from the nuclear code.

1. 17a.

   Table 2:  The Vertebrate Mitochondrial Code, differences from the standard code.  Each line gives a codon (a three-letter word in the genetic code), its meaning in this code, then its meaning in the standard code.  “AGA Ter Arg R”.  “AGG Ter Arg R”.  “AUA Met M Ile I”.  “UGA Trp W Ter *”. ↩

2. 17b.

   Table 2:  Comments printed below the table:  “at least in humans it has now been shown that AGA and AGG sequences are not recognized as termination codons. A -1 mitoribosome frameshift occurs at the AGA and AGG codons predicted to terminate the CO1 and ND6 ORFs, and consequently both ORFs terminate in the standard UAG codon (Temperley et al. 2010).” ↩

Brown, Wesley M. et al.  “Rapid evolution of animal mitochondrial DNA.”  Proceedings of the National Academy of Sciences 76(4):1967–1971.  1979.  A laboratory study that cut mitochondrial DNA from four primate species (guenon, baboon, rhesus macaque and human) with 11 restriction enzymes, which cut DNA at fixed sequences, and compared the resulting maps.  It estimated that mitochondrial DNA changes at about 0.02 substitutions per base pair per million years, roughly ten times faster than single-copy nuclear DNA.  The article traces to it the "5–10" figure that John Avise repeats.

1. 18a.

   Page 1967:  Abstract.  “The rate of evolution of the mitochondrial genome appears to exceed that of the single-copy fraction of the nuclear genome by a factor of about 10.”  Page 1969:  “This implies that mtDNA evolves 5 to 10 times faster than single-copy nuclear DNA.” ↩

Árnadóttir, Erla R. et al.  “The rate and nature of mitochondrial DNA mutations in human pedigrees.”  Cell 187(15):3904–3918.e8.  2024.  A genome study of 64,806 living Icelanders from 2,548 maternal lines, which followed 8,199 mitochondrial DNA mutations through 116,663 transmissions from mother to child.  It found that the control region of the mitochondrial genome mutates unusually often, and that only about 3 units of mtDNA reach a child from its mother, so most new mutations are lost before they spread.  The article uses it for the high pedigree rate, which it sets against the rate from family-tree comparisons, and for the bottleneck.

1. 19a.

   Section Introduction:  “The rate of mtDNA mutation typically cited in the literature is around 20 times higher than that of the nuclear genome, based on phylogenetic inference from differences between sequences with a known divergence time.” ↩

2. 19b.

   Section Results: Mutation rates by subregions and nucleotides:  “As expected, our rate estimates are substantially higher than those from phylogenetic studies: 1.25–1.70 × 10 −8 mut/bp/year for the coding region”. ↩

3. 19c.

   Section Results: Mutation rates by subregions and nucleotides:  “We identified 8,199 mutations at 3,499 positions, leading to a mutation rate estimate for the coding region of 2.87 × 10 −6 mut/bp/gen (95% CI 2.79–2.95 × 10 −6 ) or 9.79 × 10 −8 mut/bp/year”.  Arithmetic from the figures: 2.87 × 10⁻⁶ ÷ 9.79 × 10⁻⁸ = 29.3 years per generation. ↩

4. 19d.

   Section Results:  “To this end, we used a minimum read frequency threshold of 0.05 for detecting mutations in individuals, requiring at least one carrier to have a read frequency of ≥0.1”. ↩

5. 19e.

   Section Introduction:  “Selection against viable, deleterious mtDNA mutations observed in pedigrees, but too short-lived to be found on the internal branches of phylogenies, provides another important reason why mutation rates estimated from pedigrees are expected to be higher than those derived from phylogenetic inference.” ↩

6. 19f.

   Section Summary:  “Finally, we show that the fate of new mutations is determined by a drastic germline bottleneck, amounting to an average of 3 mtDNA units effectively transmitted from mother to child.” ↩

7. 19g.

   Section Introduction:  “There remains some uncertainty about the size of the bottleneck, with estimates of the effective number of mtDNA molecules passed on from mother to child ranging from 2 to 32”. ↩

Kong, Augustine et al.  “Rate of de novo mutations and the importance of father's age to disease risk.”  Nature 488(7412):471–475.  2012.  A genome study of 78 Icelandic families, each a mother, a father and a child, in which every genome was sequenced to high coverage so that new mutations, present in the child and in neither parent, could be counted.  It found an average of 1.20×10⁻⁸ new mutations per nucleotide per generation, and that each year of the father's age at conception adds about two mutations to the child.  The article uses its figure as the nuclear rate against which the mitochondrial rates are compared.

1. 20a.

   Section Abstract:  “Here we show that in our samples, with an average father’s age of 29.7, the average de novo mutation rate is 1.20×10−8 per nucleotide per generation.” ↩

Havird, Justin C., and Daniel B. Sloan.  “The Roles of Mutation, Selection, and Expression in Determining Relative Rates of Evolution in Mitochondrial versus Nuclear Genomes.”  Molecular Biology and Evolution 33(12):3042–3053.  2016.  A comparative analysis of how mitochondrial and nuclear genes change across many eukaryote lineages, including animals, plants and fungi, by two researchers at Colorado State University.  It found that how strongly selection constrains mitochondrial and nuclear genes varies widely between groups, measured as a ratio that runs from 0.19 in carnivores to more than 2 in many flowering plants.  The article uses it to qualify the claim that plant mitochondrial DNA is slow and animal mitochondrial DNA is fast.

1. 21a.

   Section Introduction:  “The mt genomes of angiosperms also fundamentally differ from those of bilaterian animals in that they have much lower rates of nucleotide substitution than the nuclear genome (Wolfe et al. 1987), which may be due to mt recombination/repair mechanisms found in plants but not animals (Christensen 2014). However, the slow-plant/fast-animal dichotomy of mt evolution is a gross oversimplification.” ↩

Allen, John F.  “The function of genomes in bioenergetic organelles.”  Philosophical Transactions of the Royal Society of London B 358:19–38.  2003.  A review that sets out a hypothesis, called CORR, for why mitochondria and chloroplasts keep some genes of their own, and tests the idea against competing views and the evidence.  The article uses it as the source of the hypothesis and of the caution about it.

1. 22a.

   Page 19:  Abstract.  “Redox control of synthesis de novo is put forward as the common property of those proteins that must be encoded and synthesized within mitochondria and chloroplasts. This testable hypothesis is termed CORR, for co-location for redox regulation.” ↩

2. 22b.

   Page 23:  “Mitochondrial departures from the ‘universal’ genetic code ... may be of selective value in that a barrier is thereby put in place to expression of genes in the ‘wrong’ compartment. This barrier is unlikely to be insuperable”. ↩

3. 22c.

   Page 37:  Thomas Cavalier-Smith, speaking in the printed discussion that follows the paper.  “there is a lot more haphazardness, and evolutionary accidents and contingency than a straight functional interpretation”. ↩

Bender, Aline et al.  “Adaptive antioxidant methionine accumulation in respiratory chain complexes explains the use of a deviant genetic code in mitochondria.”  Proceedings of the National Academy of Sciences 105(43):16496–16501.  2008.  A laboratory study that gives direct experimental evidence, in living cells, that methionine (an amino acid) building up inside the membrane acts as an antioxidant, a substance that blocks damage from oxygen-based chemicals, and protects the cells.  Its title argues that this explains why mitochondria use a deviant genetic code, one that reads some codons differently from the standard code.  The article uses it for the one change to that code that has a proposed function.

1. 23a.

   Section Abstract, page 16496:  “we present direct experimental evidence that intramembrane accumulation of methionine exhibits antioxidant and cytoprotective properties in living cells.” ↩

von Heijne, Gunnar.  “Why mitochondria need a genome.”  FEBS Letters 198:1–4.  1986.  A four-page paper from 1986 in which the author suggests a reason mitochondria keep some genes of their own.  The article uses it for the proposal that the cell's protein-export machinery limits which mitochondrial genes can be moved to the nucleus.

1. 24a.

   Section Abstract, page 1:  “It is suggested that the machinery for co-translational protein export across the endoplasmic reticulum membrane sets strict limits on the kinds of protein-coding genes that can be successfully transferred from the mitochondrial to the nuclear genome. This hypothesis is in perfect agreement with the pattern of mitochondrially vs nuclearly encoded mitochondrial proteins found in species such as man, mouse, and Xenopus.” ↩

Oca-Cossio, Jose et al.  “Limitations of Allotopic Expression of Mitochondrial Genes in Mammalian Cells.”  Genetics 165(2):707–720.  2003.  A laboratory study that tried to move three mitochondrial genes into the nucleus, recoding each for nuclear use, giving each a mitochondrial address tag, and expressing them in two kinds of cultured cells, COS-7 and HeLa.  Only ATPase8 was correctly expressed and sent to the mitochondria, while the other two clumped together in fiber-like structures, and expressing two of them caused a loss of mitochondrial membrane potential, which can lead to cell death.  The article uses it as the test that found only some mitochondrial genes can be replaced in the nucleus.

1. 25a.

   Section Abstract, page 707:  “In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria."  …  "Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically.” ↩

Allen, John F.  “Why chloroplasts and mitochondria retain their own genomes and genetic systems: Colocation for redox regulation of gene expression.”  Proceedings of the National Academy of Sciences 112(33):10231–10238.  2015.  A review by John Allen of University College London of the evidence for his CoRR hypothesis, which says a gene stays inside its organelle so that the organelle can adjust the gene's output to its own redox state, the balance of electrons in its energy chain.  It reviews experiments rather than reporting a new one.  The article uses it for Allen's own statement of the hypothesis, the test that would disprove it, and his doubt about mitochondria.

1. 26a.

   Page 10231:  Abstract.  “Colocation of gene and gene product for redox regulation of gene expression (CoRR) is a hypothesis in agreement with the results of a variety of experiments designed to test it and which seem to have no other satisfactory explanation.” ↩

2. 26b.

   Page 10234:  “In view of the experimental progress with chloroplasts, what can be said concerning redox control of gene expression in mitochondria?” ↩

3. 26c.

   Page 10235:  “There seems so far to be no example of a chemiosmotically functional subcellular compartment that does not carry genes for some of its own components. One example would suffice to disprove CoRR.” ↩

Johnston, Iain G., and Ben P. Williams.  “Evolutionary Inference across Eukaryotes Identifies Specific Pressures Favoring Mitochondrial Gene Retention.”  Cell Systems 2(2):101–111.  2016.  A computer study that infers how mitochondrial genes were lost over evolution, using 2015 complete mitochondrial genomes from across the eukaryotes, the organisms whose cells have a nucleus.  It found that the proteins forming the structural cores of the electron transport chain are the ones most often kept in mitochondria, and that high GC content (a high share of the letters G and C in the DNA) together with high hydrophobicity (protein parts that avoid water) is needed to explain which genes are kept.  The article uses it for the finding that hydrophobicity, which makes proteins hard to import from outside the mitochondria, helps explain which genes stay.

1. 27a.

   Section Summary, page 101:  “Using 2015 complete mitochondrial genomes, we inferred evolutionary trajectories of mtDNA gene loss across the eukaryotic tree of life. We find that proteins comprising the structural cores of the electron transport chain are preferentially encoded within mitochondrial genomes across eukaryotes. A combination of high GC content and high protein hydrophobicity is required to explain patterns of mtDNA gene retention”. ↩

2. 27b.

   Page 105:  CoRR stands for colocalization for redox regulation, Allen's hypothesis about why key organellar genes are retained.  “This link between biophysical and evolutionary features of mitochondrial genes is consistent with the CoRR hypothesis, supporting its applicability to organellar genomes of multiple and diverse taxa across the tree of life.” ↩

Perales-Clemente, Ester et al.  “Allotopic expression of mitochondrial-encoded genes in mammals: achieved goal, undemonstrated mechanism or impossible task?”  Nucleic Acids Research 39(1):225–234.  2011.  A laboratory study that tried to replace a missing mitochondrial gene, mt-Nd6, in mouse cells by giving them a recoded copy of the gene in the nucleus, so that the cell makes the protein outside the mitochondria and imports it.  The cells appeared rescued, but the authors found that the protein was not inside the mitochondria and that the surviving clones were revertants, cells whose mt-Nd6 gene had mutated back to the normal sequence.  The article uses it as an early warning that rescue experiments can be misread.

1. 28a.

   Section Abstract:  “Here, we show that reliance on partial rescue alone can lead to a false conclusion of successful allotopic expression."  …  "However, careful examination of transfected cells showed that the allotopically-expressed protein was not internalized in mitochondria and that the selected clones were in fact revertants for the mt-Nd6 mutation.” ↩

Newman, Nancy J. et al.  “Randomized trial of bilateral gene therapy injection for m.11778G>A MT-ND4 Leber optic neuropathy.”  Brain 146(4):1328–1341.  2023.  A randomized trial, in which patients were assigned to treatment by chance, of a gene therapy for Leber hereditary optic neuropathy (LHON), an inherited disease that damages the optic nerve.  It gave 98 patients an injection in both eyes, or an injection in one eye and a placebo (a dummy injection) in the other.  GenSight Biologics, the sponsor, funded the study, and several of the authors are employed by it.  The article uses it as the trial of the ND4 gene therapy that missed its main measure.

1. 29a.

   Section Introduction:  “The m.11778G>A mutation is the most common LHON mutation, accounting for about 70% of LHON in North America and Europe.” ↩

2. 29b.

   Section Introduction:  “Lenadogene nolparvovec is a replication-defective single-stranded DNA recombinant adeno-associated virus vector 2, serotype 2 (rAAV2/2), containing a codon-modified complementary DNA (cDNA) that encodes for the human wild-type ND4 protein".  …  "The construct includes the cis-acting elements of the human cytochrome c oxidase 10 (COX10) mtRNA (MTS in 5′ of the cDNA, and 3′ UTR at the 3′ end of the cDNA) ensuring the efficient delivery of the corresponding hybrid mRNA to the mitochondrial surface.” ↩

3. 29c.

   Section Results:  BCVA means best-corrected visual acuity, the sharpest sight a patient reaches with glasses or lenses.  “The least squares (LS) mean difference in the change of BCVA between these two treatment arms at 1.5 years was −0.05 LogMAR [P = 0.6080, analysis of covariance (ANCOVA)]. The primary end point, defined as a difference of at least −0.3 LogMAR, was therefore not met.” ↩

4. 29d.

   Section Discussion:  The authors, several of them employed by GenSight Biologics, the sponsor of these studies, write:  “The lack of a patient placebo-controlled arm in REFLECT and previous studies necessitates the indirect comparison of outcomes with published natural history data.” ↩

Yu-Wai-Man, Patrick et al.  “Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy.”  Science Translational Medicine 12(573):eaaz7423.  2020.  A clinical trial report on the same ND4 gene therapy, in which one eye was injected and the other was given a sham (fake) injection.  Both eyes improved, but the primary end point, a comparison of the change in sight between the two groups, was not met.  The article uses it for the authors' argument that DNA from the virus crossed to the uninjected eye, a plausible reason for the unexpected improvement in both eyes.

1. 30a.

   Section Abstract:  “At week 96, rAAV2/2-ND4-treated eyes showed a mean improvement in best-corrected visual acuity (BCVA) of -0.308 LogMAR (+15 ETDRS letters). A mean improvement of -0.259 LogMAR (+13 ETDRS letters) was observed in the sham-treated eyes. Consequently, the primary end point, defined as the difference in the change in BCVA from baseline to week 48 between the two treatment groups, was not met (P = 0.894)."  …  "Evidence of transfer of viral vector DNA from the injected eye to the anterior segment, retina, and optic nerve of the contralateral noninjected eye supports a plausible mechanistic explanation for the unexpected bilateral improvement in visual function after unilateral injection.” ↩

Yu-Wai-Man, Patrick et al.  “Five-Year Outcomes of Lenadogene Nolparvovec Gene Therapy in Leber Hereditary Optic Neuropathy.”  JAMA Ophthalmology 143(2):99.  2025.  A five-year follow-up of the RESTORE trial, whose patients had one eye treated with lenadogene nolparvovec, published online on 19 December 2024.  GenSight Biologics, the sponsor, funded the study and took part in its design, data analysis and manuscript preparation.  The article uses it as the sponsor's report of lasting improvement, set against limits the authors admit.

1. 31a.

   Section Abstract, Conclusions and Relevance:  “In this analysis of the RESTORE trial, follow-up of patients with LHON due to the MT-ND4 gene variant unilaterally treated with lenadogene nolparvovec demonstrated a sustained bilateral improvement in BCVA and a good safety profile up to 5 years after treatment.” ↩

2. 31b.

   Section Discussion, limitations:  “In turn, one explanation for improvement from the nadir could be regression to the mean, that is, the BCVA improved from 1 BCVA obtained at 1 time, which was low due to poor effort or fixation. Another major limitation of this study includes the lack of a control group of untreated patients raising difficulties in interpreting the actual treatment effect.” ↩

Elliott, Hannah R. et al.  “Pathogenic mitochondrial DNA mutations are common in the general population.”  American Journal of Human Genetics 83(2):254–260.  2008.  A study of 3,168 cord-blood samples from newborns in north Cumbria, England, tested for ten mutations often found in people with mitochondrial disease, and compared each with the mother's blood.  It found such a mutation in 15 babies (0.54%), and the authors conclude that at least one in 200 healthy people carries a mutation that could cause disease in the children of female carriers.  The article uses it as its figure for how often healthy people carry a disease-causing mutation.

1. 32a.

   Section Abstract:  “We determined the frequency of ten mitochondrial point mutations in 3168 neonatal-cord-blood samples from sequential live births”.  “In conclusion, at least one in 200 healthy humans harbors a pathogenic mtDNA mutation that potentially causes disease in the offspring of female carriers.” ↩

Stewart, James B. et al.  “Strong Purifying Selection in Transmission of Mammalian Mitochondrial DNA.”  PLoS Biology 6(1):e10.  2008.  A research paper on how mammalian mitochondrial DNA is passed on from one generation to the next.  It reports a rapid and strong elimination of changes that alter the proteins made from mitochondrial genes, the hallmark of purifying selection.  The article quotes its statement of the expected build-up of harmful mutations in a genome passed on without sex, the argument known as Muller's Ratchet.

1. 33a.

   Section Author Summary:  “This asexual mode of transmission, together with a high rate of mutation, should eventually lead to the accumulation of numerous deleterious mtDNA mutations and a “mutational meltdown” (a phenomenon know as Muller's Ratchet).”  "know" is in the original. ↩

2. 33b.

   Section Abstract:  “We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection.” ↩

Gemmell, Neil J. et al.  “Mother's curse: the effect of mtDNA on individual fitness and population viability.”  Trends in Ecology & Evolution 19(5):238–244.  2004.  A paper on how mitochondrial DNA affects the fitness of individuals and the survival of populations.  Its abstract says that harmful mutations which affect only males escape natural selection, because mitochondria pass through mothers and so can spread through a population when the mutations do no harm to females.  The article uses it for the "mother's curse" idea, that mutations which harm only males are not removed by selection.

1. 34a.

   Page 238, section Abstract:  “Ironically, deleterious mtDNA mutations that affect only males, such as those that impair sperm function, will not be subject to natural selection because mitochondria are generally maternally inherited and could reach high frequencies in populations if the mutations are not disadvantageous in females.” ↩

Rana, Fazale.  “Mitochondria's Deviant Genetic Code: Evolution or Creation?”  Reasons to Believe.  Apr 11, 2018.  A web article by Fazale Rana of Reasons to Believe, on why mitochondria use a genetic code that differs from the standard one.  It explains a study by German biochemists, Aline Bender and colleagues, which found that swapping the isoleucine codon for methionine protects the inner-membrane proteins of mitochondria from oxidative damage.  The article quotes Rana's conclusion from this study that a creation model has good reasons for mitochondrial biochemistry.

1. 35a.

   Section Closing paragraph:  “From a creation model vantage point, there are good reasons why things are the way they are regarding mitochondrial biochemistry.” ↩

Rana, Fazale.  “Why Mitochondria Make My List of Best Biological Designs.”  Reasons to Believe.  May 1, 2019.  A web article by Fazale Rana of Reasons to Believe, arguing that mitochondria are among the best-designed features of living things.  It builds on Nick Lane's argument that bacteria and archaea cannot grow complex because of energy limits, and that mitochondria lift that limit, using Lane's measure of available energy per gene (AEG).  The article quotes it for Rana's claim that the small mitochondrial genome is itself one of the features that makes the design good.

1. 36a.

   Section Bioenergetic Freedom for Eukaryotic Cells:  “To summarize, mitochondria’s small genome plus its prodigious ATP output are the keys to eukaryotic cells’ large AEG.” ↩